This grant will examine the hypothesis that receptor-mediated endocytosis limits cellular responses to GRP both by receptor internalization and by intracellular lysosomal destruction of internalized GRP. The second hypothesis is that pH sensitive endosomal dissociation of ligand regulates receptor recycling and resensitization and thereby regulates responsiveness to activation of G protein coupled receptors (GPCRs). The local concentration of hormones or neuropeptides, and the number of responsive cell surface receptors, regulate receptor-mediated responses. The general phenomenon of receptor desensitization and resensitization is vital to all processes that are regulated by receptors. Just as it is necessary to have an intracellular signal transduction system to produce responses to extracellular ligands, it is also necessary to have mechanisms to terminate the responses or to maintain them. Using GRP receptor as a general model for gastrointestinal GPCRs, we will define the processes of endosomal uptake of GRP receptors, pH dependent intraendosomal dissociation of GRP lysosomal degradation of endocytosed GRP, and receptor recycling and reactivation by: a) using the rate of GRP degradation to measure turnover rates of functional GRP receptors; b) determining the role of acidification of endosomes and lysosomes for dissociation and enzymatic degradation of ligand and for recycling and resensitization of the receptor; c) determining the roles of phosphorylation and dephosphorylation in receptor desensitization and resensitization and characterizing the intracellular sites and pH at which these occur; and d) identifying interactions between receptors tagged with green fluorescent proteins, Cy3 labeled GRP, and other molecules that are colocalized at different stages of endocytosis and determine the effects of cytoskeletal elements and of colocalized substances on endocytosis and recycling. We also plan to characterize GRP receptor recycling and resensitization in cells that normally express GRP receptors: gastric G cells, Swiss 3T3 cells, and small cell lung carcinoma line NCI-H345 cells.